Investigation of the roles of CheD in the Bacillus subtilis chemotactic signal transduction

Abstract The two-component based chemotaxis signal transduction system allows flagellated bacteria to sense their surrounding chemical environment and move towards more favorable conditions. The attractant signals can be sensed by transmembrane chemoreceptors, and then transmitted to the histidine kinase CheA. Once activated, CheA interacts with the response regulator CheY through phosphorelay, which causes a change in the rotation of the flagella. The direction of flagella rotation determines whether a cell swims straight or just tumbles. Cells also need adaptation to respond to a change in chemical concentrations, and return to their prestimulated level. Adaptation in the B. subtilis chemotaxis system is achieved by three coordinated systems: the methylation system, the CheC/CheD/CheY-p system and the CheV system. CheD, the previously identified receptor deamidase, was shown to be critical to the ability of B. subtilis to perform chemotaxis and is the main focus of this study. This study started from characterization of the enzymatic mechanism of CheD. Results showed that CheD deamidase uses a cysteine hydrolase mechanism. The catalytic triad consisting of Cys33-His50-Thr27, and Ser27 is essential for receptor recognition and binding. In addition, in this study CheC was found to inhibit CheD’s deamidase activity. Through mutant screening, Phe102 on CheD was found to be the essential site to interact with CheC. Furthermore, the CheD/CheC interaction is necessary for the robust chemotaxis in vivo as demonstrated by the cheD (F102E) mutant, which lacks the ability to swim on swarm plates. Despite its deamidase activity, we hypothesized that CheD’s main role is its involvement in the CheD-CheC-CheY-p negative feedback pathway during adaptation. In particular, CheD is likely to help stabilize the transient kinase-activating state through binding to receptors. When CheY-p level is increased, CheC-CheY-p complex may attract CheD away from receptors. In this study, CheC-CheD binding kinetics with CheY or CheYp presence was successfully obtained by a series of SPR experiments. The increased affinity of CheD for CheC in presence of CheYp but not CheY makes likely the hypothesis that CheC-CheD-CheY interact as part of a negative feedback pathway during adaptation. Last, the interaction between CheD and chemoreceptor McpC was studied in order to better understand the role of CheD in adaptation. Results showed that Q304 and Q305 on McpC are essential to recruit CheD. Additionally, the reduced levels of CheD in mcpC (Q304A) or (Q305A) mutants suggested that the dynamic interaction between CheD and receptors is vital to maintain the normal CheD level. These findings suggest more complicated roles of CheD than its previously identified function as a receptor deamidase, and will lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction system.

Diet and Probiotics during Pregnancy - Endorsing developmental Programming

Maternal obesity has been shown to increase the risk for adverse reproductive health outcomes such as gestational diabetes, hypertension, and preeclampsia. Moreover, several studies have indicated that overnutrition and maternal obesity adversely program the development of offspring by predisposing them to obesity and other chronic diseases later in life. The exact molecular mechanisms leading to developmental programming are not known, but it has recently been suggested that obesity-related low-grade inflammation, gut microbiota and epigenetic gene regulation (in particularly DNA methylation) participate in the developmental programming phenomenon. The aim of this thesis was to evaluate the effect of diet, dietary counseling and probiotic intervention during pregnancy in endorsing favorable developmental programming. The study population consisted of 256 mother-child pairs participating in a prospective, double-blinded dietary counselling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) study. Further overweight women were recruited from maternal welfare clinics in the area of Southwest Finland and from the prenatal outpatient clinic at Turku University Hospital. Dietary counseling was aimed to modify women’s dietary intake to comply with the recommended intake for pregnant women. Specifically, counseling aimed to affect the type of fat consumed and to increase the amount of fiber in the women’s diets. Leptin concentration was used as a marker for obesity-related low-grade inflammation, antioxidant vitamin status as an efficiency marker for dietary counselling and epigenetic DNA methylation of obesity related genes as a marker for probiotics influence. Results revealed that dietary intake may modify obesity-associated low-grade inflammation as measured by serum leptin concentration. Specifically, dietary fiber intake may lower leptin concentration in women, whereas the intakes of saturated fatty acids and sucrose have an opposite effect. Neither dietary counselling nor probiotic intervention modified leptin concentration in women, but probiotics tended to increase children’s leptin concentration. Dietary counseling was an efficient tool for improving antioxidant vitamin intake in women, which was reflected in the breast milk vitamin concentration. Probiotic intervention affected DNA methylation of dozens of obesity and weight gain related genes both in women and their children. Altogether these results indicate that dietary components, dietary counseling and probiotic supplementation during pregnancy may modify the intrauterine environment towards favorable developmental programming.

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

Identification and characterisation of telomere regulatory and signalling pathways after induction of telomere dysfunction

Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.

Analiza proteomiczna kompleksów tworzonych przez białko hDPY30 w komórkach ludzkich

Wydział Biologii

Targeting the serrated pathway of colorectal cancer with mutation in BRAF

A recently acknowledged morphological pathway to colorectal cancer originates from precursor polyps with a serrated appearance due to branching and folding of the colon epithelium. This serrated origin accounts for up to 30% of all colorectal tumors but these are heterogeneous regarding molecular characteristics and patient outcome. Here we review the current knowledge about the classification of this tumor subtype and its association with five key features: mutation status of the BRAF or KRAS genes, the CpG island methylation phenotype, microsatellite instability, immune cell infiltration, and overexpression of GTPase RAC1b. Subsequently, available therapeutic approaches for targeting these molecular characteristics are presented and critically discussed.

Análise de expressão de genes da família SETD em leucemia linfocítica crônica

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

DNA demethylation during Chrysanthemum floral transition following short-day treatment

Background: Analytical techniques such as methylation-sensitive amplification polymorphism and high-performance liquid chromatography were used to detect variation in DNA methylation of mature Chrysanthemum leaves during the floral transition induced by short-day (SD) treatment. Results: For both early- and late-flowering cultivars, the time from the date of planting to the appearance of the capitulum bud and early blooming were significantly shorter than those of the control. The capitulum development of the early-flowering cultivar was significantly accelerated compared to the control, unlike the late-flowering cultivar. The DNA methylation percentage of leaves was significantly altered during flower development. For the early-flowering cultivar, DNA methylation was 42.2–51.3% before the capitulum bud appeared and 30.5–44.5% after. The respective DNA methylation percentages for the late-flowering cultivar were 43.5–56% and 37.2–44.9%. Conclusions: The DNA methylation percentage of Chrysanthemum leaves decreased significantly during floral development. The decline in DNA methylation was elevated in the early-flowering cultivar compared with the late-flowering cultivar.

Dynamique de la variation génétique et épigénétique chez un vertébré kleptogène

La variation phénotypique est essentielle à la persistance des organismes dans le temps ainsi qu’à la colonisation de nouveaux habitats. Les principales sources de variation phénotypique sont la génétique et l'épigénétique. L'épigénétique a été proposé comme un atout important pour les organismes asexués pour compenser le manque de diversité génétique. L'objectif de cette étude est d'évaluer si l’absence de variation génétique est compensée par l'épigénétique en comparant les profils de méthylation d’individus gynogènes et kleptogènes des hybrides de salamandre à points bleus. Les individus échantillonnés s’organisent en cinq groupes génétiquement différenciés, provenant du même haplome paternel A. jeffersonianum. Deux des cinq groupes sont exclusivement gynogènes, pour des raisons écologiques ou génomiques. Les trois autres groupes sont formés d’individus parfois kleptogènes, car ils présentent une variation génétique plus élevée au sein d’un site qu’entre les sites, en plus de porter des allèles très divergents par rapport à la distribution globale des allèles hybrides, trouvés en haute fréquence dans les populations sympatriques de A. laterale. Les patrons épigénétiques sont variables et distincts entre les cinq groupes génétiques. Les groupes gynogènes sont les seuls à présenter un effet environnemental significatif sur leurs patrons épigénétiques, suggérant que ces individus clonaux doivent être en mesure de maximiser leur potentiel de variation épigénétique pour faire face à des variations environnementales.

Resposta de Chromobacterium Violaceum cultivada em alta concentração de ferro

The Chromobacterium violaceum is a β-proteobacterium Gram-negative widely found in tropical and subtropical regions, whose genome was sequenced in 2003 showing great metabolic versatility and biotechnological and pharmaceutical potential. Given the large number of ORFs related to iron metabolism described in the genome of C. violaceum, the importance of this metal for various biological processes and due to lack of data about the consequences of excess of iron in free-living organisms, it is important to study the response mechanism of this bacterium in a culture filled with iron. Previous work showed that C. violaceum is resistant to high concentrations of this metal, but has not yet been described the mechanism which is used to this survival. Thus, to elucidate the response of C. violaceum cultured in high concentrations of iron and expecting to obtain candidate genes for use in bioremediation processes, this study used a shotgun proteomics approach and systems biology to assess the response of C. violaceum grown in the presence and absence of 9 mM of iron. The analysis identified 531 proteins, being 71 exclusively expressed by the bacteria grown in the presence of the metal and 100 just in the control condition. The increase in expression of proteins related to the TCA cycle possibly represents a metabolic reprogramming of the bacteria caused by high concentration of iron in the medium. Moreover, we observed an increase in the activity assay of superoxide dismutase and catalase as well as in Total Antioxidant Activity assay, suggesting that the metal is inducing oxidative stress in C. violaceum that increases the levels of violacein and antioxidant enzymes to better adapt to the emerging conditions. Are also part of the adaptive response changes in expression of proteins related to transport, including iron, as well as an increased expression of proteins related to chemotaxis response, which would lead the bacteria to change the direction of its movement away from the metal. Systems Biology results, also suggest a metabolic reprogramming with mechanisms coordinated by bottleneck proteins involved in transcription (GreA), energy metabolism (Rpe and TpiA) and methylation (AhcY)

Régulation épigénétique de la défense antioxydante et de l'Angiopoietin-like 2 dans le contexte du vieillissement et des maladies cardiovasculaires

Suite à l’exposition à des facteurs de risque incluant la malnutrition, la dyslipidémie, la sédentarité et les désordres métaboliques, les maladies cardiovasculaires (MCV) sont caractérisées par un état pro-oxydant et pro-inflammatoire, et une dérégulation de l’expression de divers facteurs responsables de l’homéostasie de l’environnement rédox et inflammatoire. L’implication d’enzymes antioxydantes telles que les superoxyde dismutases (SOD) et les glutathion peroxydases (Gpx), ainsi que la contribution de médiateurs pro-inflammatoires tels que l’angiopoietin-like 2 (Angptl2) ont été rapportées dans le cadre des MCV. Toutefois, les mécanismes moléculaires sensibles aux facteurs de risque et menant au développement des MCV sont peu connus. L’épigénétique est un mécanisme de régulation de l’expression génique sensible aux stimuli extracellulaires et pourrait donc contribuer au développement des MCV. La méthylation de l’ADN est un des mécanismes épigénétiques pouvant varier tant de manière gène-spécifique qu’à l’échelle génomique, et la conséquence de tels changements sur l’expression des gènes ciblés dépend du site de méthylation. Puisqu’il a été démontré que des variations au niveau de la méthylation de l’ADN peuvent être associées à divers contextes pathologiques incluant les MCV, le but de nos travaux était d’étudier le lien entre la méthylation de gènes antioxydants et pro-inflammatoires avec leurs répercussions fonctionnelles biologiques en présence de facteurs de risques associés aux MCV, tels que le vieillissement, la dyslipidémie et la sédentarité. Dans la première étude, nous avons observé que dans l’artère fémorale de souris vieillissantes, la méthylation au niveau du promoteur du gène Sod2, codant pour l’enzyme antioxydante superoxyde dismutase de type 2 (SOD2 ou MnSOD), diminue avec l’âge. Ceci serait associé à l’induction de l’expression de MnSOD, renforçant ainsi la défense antioxydante endogène. Le vieillissement étant associé à une accumulation de la production de radicaux libres, nous avons étudié la vasodilatation dépendante de l’endothélium qui est sensible au stress oxydant. Nous avons observé que la capacité vasodilatatrice globale a été maintenue chez les souris âgées, aux dépens d’une diminution des facteurs hyperpolarisants dérivés de l’endothélium (EDHF) et d’une contribution accentuée de la voie du monoxyde d’azote (NO). Nous avons ensuite utilisé deux approches visant à réduire les niveaux de stress oxydant in vivo, soit la supplémentation avec un antioxydant, la catéchine, et l’exposition chronique à de l’exercice physique volontaire. Ces interventions ont permis de prévenir à la fois les changements au niveau de la fonction endothéliale et de l’hypométhylation de Sod2. Cette première étude démontre donc la sensibilité de la méthylation de l’ADN à l’environnement rédox. Dans la deuxième étude, nous avons démontré une régulation de l’expression de l’enzyme antioxydante glutathion peroxydase 1 (Gpx1) en lien avec la méthylation de son gène codant, Gpx1, dans un contexte de dyslipidémie sévère. Nos résultats démontrent que dans le muscle squelettique de souris transgéniques sévèrement dyslipidémiques (LDLr-/-; hApoB+/+), Gpx1 est hyperméthylé, ce qui diminue l’expression de Gpx1 et affaiblit la défense antioxydante endogène. Chez ces souris, l’exercice physique chronique a permis d’augmenter l’expression de Gpx1 en lien avec une hypométhylation transitoire de son gène. Cette étude démontre que le stress oxydant associé à la dyslipidémie sévère altère les mécanismes de défense antioxydante, en partie via un mécanisme épigénétique. De plus, on observe également que l’exercice physique permet de renverser ces effets et peut induire des changements épigénétiques, mais de manière transitoire. La troisième étude avait pour but d’étudier la régulation de l’Angptl2, une protéine circulante pro-inflammatoire, dans le contexte des MCV. Nous avons observé que chez des patients coronariens, la concentration circulante d’Angptl2 est significativement plus élevée que chez des sujets sains et ce, en lien avec une hypométhylation de son gène, ANGPTL2, mesurée dans les leucocytes circulants. Nous sommes les premiers à démontrer qu’en réponse à l’environnement pro-inflammatoire associé à une MCV, l’expression de l’Angptl2 est stimulée par un mécanisme épigénétique. Nos études ont permis d’identifier des nouvelles régions régulatrices différentiellement méthylées situées dans les gènes impliqués dans la défense antioxydante, soit Sod2 en lien avec le vieillissement et Gpx1 en lien avec la dyslipidémie et l’exercice. Nous avons également démontré un mécanisme de régulation de l’Angptl2 dépendant de la méthylation d’ANGPTL2 et ce, pour la première fois dans un contexte de MCV. Ces observations illustrent la nature dynamique de la régulation épigénétique par la méthylation de l’ADN en réponse aux stimuli environnementaux. Nos études contribuent ainsi à la compréhension et l’identification de mécanismes moléculaires impliqués dans le développement du phénotype pathologique suite à l’exposition aux facteurs de risque, ce qui ouvre la voie à de nouvelles approches thérapeutiques.